The long term goal of these studies is to develop a detailed understanding of the mechanism by which herpes viruses replicate their DNA. This process requires a series of herpes encoded proteins and may also involve cellular proteins. The proposed studies will focus on two complexes containing 5 essential proteins - the herpes polymerase complex (UL30/UL42) and the primase-helicase complex (UL5/UL52/UL8). The specific aims of the proposal are: (1) Determine if the cellular pol 1 is required for herpes replication using both siRNA knockdown of pol 1 and a specific small molecule inhibitor of pol 1. (2) Develop a mechanistic understanding of how the interplay between polymerase activity, exonuclease activity, and UL42 enhances the fidelity of replication using a variety of kinetic and mechanistic probes (3) Elucidate the mechanistic consequences of coupled activity between the polymerase and helicase and if the helicase can displace tightly bound proteins from DNA. Both steady- state and pre-steady state kinetic approaches will be used. (4) Using purified proteins and a minicircle template that gives efficient leading and lagging strand, examine the mechanistic coupling of the leading and lagging strand replication apparatus and the effects of bound proteins on the progression of the replication fork. These studies will provide novel insights into how the different proteins functionally interact with each other and the mechanism of herpes DNA replication. PUBLIC HEALTH RELEVANCE: Herpes viruses are significant human pathogens and cause a number of potentially very severe diseases. The herpes replication apparatus is the primary target of currently used anti- herpes drugs. The proposed studies will lead to a better understanding of herpes replication, and thus could lead to the development of new and more effective drugs.